Immunization with nucleic acids has the potential to circumvent many of the problems associated with protein vaccines.
Nucleic acid vaccines are relatively inexpensive to produce, overcoming the high costs of production and difficulties in purification associated with the preparation of soluble protein antigens. Furthermore, immunization with protein antigen is associated with problems of incorrect folding and poor induction of cell-mediated immunity. In contrast, nucleic acid immunization generates correctly folded conformational determinants and maximizes the presentation of processed antigenic determinants on MHC molecules, eliciting both humoral and cell-mediated immunity. Nucleic acid vaccination therefore holds great promise for infectious disease prophylactics and immunotherapy.
The efficacy of DNA vaccination has been successfully demonstrated in animal models for a wide range of pathogens (Pardoll, 1995; Donnelly, 1997). However, there are still major practical and theoretical obstacles to overcome before the potential of these vaccines can be realized. Most animal model studies were carried out in rodents and it subsequently became clear that in larger animals, in particular primates and humans, DNA vaccination has limited efficacy (Fuller, 1997; Wang, 1998). Although DNA vaccines typically elicit good cell-mediated immunity, they elicit poor humoral immunity, leading some to conclude that nucleic acid vaccines will not replace conventional protein vaccines.
Several strategies to improve the immunogenicity of DNA vaccines and manipulate the immune response have been developed, such as the use of various viral, eukaryotic and combined promoters (Donnelly, 1997; Barnhart, 1998; Armand, 2000; Kwissa, 2000), the improvement of transgene expression (Hartikka, 1996; Yew, 1997), the addition of immunostimulatory sequences into the vector backbone which act in vivo as a T helper 1 enhancing adjuvant (Sato, 1996), or the co-expression of costimulatory molecules, such as cytokines or chemokines (Leitner, 2000).
However, none of these strategies has been particularly successful and there remains a need to increase the immunogenicity of nucleic acid vaccines and enhance humoral responses elicited by nucleic acid vaccines. An object of the invention is to provide nucleic acid vaccines which elicit good humoral responses, making them a viable alternative to conventional vaccines.
An approach suggested by Boyle et al involves targeting antigen to sites of immune induction by vaccination with DNA encoding the antigen as a CTLA-4 fusion protein (Boyle, 1998). CTLA-4 is expressed on activated T cells and binds to the surface receptor B7-1 and B7-2 of antigen-presenting cells (APCs), which are potent initiators of immune responses (Linsley, 1990). Deliyannis et al demonstrated that this approach enhanced the speed and magnitude of the immune response against a viral antigen in mice. Vaccinated mice were found to have significantly reduced viral titres when faced with viral challenge (Deliyannis et al, 2000). Chaplin et al found that targeting improved the efficacy of a DNA vaccine against Corynebacterium in sheep (Chaplin et al, 1999).
These workers used vaccination vectors comprising a plasmid encoding an antigen fused to a CTLA-4-human immunoglobulin structure. High avidity binding of CTLA-4 to its receptor requires CTLA-4 dimerization (Greene, 1996), and this dimerization is also critical for the observed enhancement of the immune response (Lew, 2000). As a dimerization moiety, the authors used either the hinge and heavy chain constant domains of human IgG1, or only the hinge regions of human IgG3.
However, this approach also results in antigen dimerization which can have negative effects on the quality of the immune response. For example, monomers but not dimers of recombinant HCV glycoprotein E2, expressed in chinese hamster ovary cells, have the biological activity required to elicit a good antibody response (Heile, 2000). Recombinant E2 protein expressed in mammalian cells tends to form aggregates, which are stabilised by disulphide bonds. It is likely that such aggregates will also form when E2 is forced to be in close vicinity with a copy of itself in the context of a dimerized fusion protein. Thus, an object of the invention is the provision of more sophisticated nucleic acid vectors which allow the antigen to be targeted to the preferred cells in a monomeric state.
Similarly, there is a need for nucleic acid vectors that would allow the homodimerization or heterodimerization of the targeting moiety whilst allowing antigen heterodimerization. Taking HCV as an example again, E2 heterodimerizes with E1 in vitro. When vaccinated with recombinant E1-E2 heterodimers, chimpanzees develop high titres of anti-E2 antibodies in serum and are completely protected from subsequent challenge with the homologous virus (Choo, 1994; Rosa, 1996). An object of the invention is to circumvent the inherent difficulties associated with producing E1-E2 heterodimers by providing a nucleic acid vaccine encoding an E1-E2 heterodimer such that the heterodimer is targeted to APCs. A further object of the invention is to provide nucleic acid vaccines which stimulate effective cell-mediated and humoral immune responses against more than one antigen
Monoclonal antibodies are invaluable research tools for various techniques such as ELISA, immunoblots, immunohistology and cytofluorimetry and are often the key to the characterization of protein structure, function and purification. They permit efficient purification by affinity chromatography and allow functional characterization by blocking binding or active sites. Traditionally, the production of specific polyclonal and monoclonal antibodies has required the purification and injection of significant amounts of protein from tissues or fluids for immunization, or the injection of mammalian cell clones expressing certain markers, followed by extensive screening procedures. It is now possible to obtain information about a protein from its cloned sequence without ever having purified it, and recombinant forms of the protein can be expressed and purified from cells or supernatent in various systems. However, this remains a difficult undertaking and the process of obtaining recombinant protein in native conformation in sufficient yield and purity for immunization and screening still remains a limiting step in the path towards obtaining antibodies. Immunizing mice or other small animals with nucleic acid encoding the protein has been suggested as a way of overcoming these problems (Ulvieri, 1996). However, current nucleic acid immunization vectors do not provide sufficient yields to make this method viable. An object of the invention is the provision of nucleic acid vectors which allow increased antibody yields to be obtained.